However, the role of c-Jun in metastasis of human breast cancer in vivo is currently unknown.c-Jun/AP-1 has been linked to invasive properties of aggressive breast cancer. Recently, it has been reported that overexpression of c-Jun in breast cancer cell line MCF-7 resulted in increased AP-1 activity, motility and invasiveness of the cells in vitro and in nude mice.To further investigate the direct involvement of c-Jun in tumorigenesis and metastasis, in the present study, the effects of c-Jun overexpression were studied in both Ectopic overexpression of c-Jun promoted the growth of MCF-7 cells and resulted in a significant increase in the percentage of cells in S phase and increased motility and invasiveness. Introduction of c-Jun gene alone into weakly invasive MCF-7 cells resulted in the transfected cells capable of metastasizing to the nude mouse liver following tail vein injection.in vivo liver metastasis of MCF-7/c-Jun cells. The results provide further evidence that c-Jun is involved in the metastasis of breast cancer. The finding also opens an opportunity for development of anti-c-Jun strategies in breast cancer therapy.The present study confirms that overexpression of c-Jun contributes to a more invasive phenotype in MCF-7 cells. It indicates an interesting relationship between c-Jun expression and increased property of adhesion, migration and  In vitro studies have demonstrated that Fos/Jun heterodimers are more stable and efficient in driving transcriptional activation than a Jun/Jun homodimer  and centrifuged at 10 000 × g at 4°C for 10 minutes. Supernatants were transferred to new tubes. Samples were denatured by being heated to 100°C prior to fractionation onto SDS-PAGE gels. After transfer to nitrocellulose membranes, filters were blocked for 1 hour in blocking buffer  and then incubated for 1 hour with the primary antibody against c-Jun  diluted in blocking buffer. After being washed with TBS , filters were incubated with horseradish peroxidase-conjugated secondary antibody (Beijing Zhongshan Golden Bridge Biotechnology Co. LTD) for 30 minutes and bands detected by SuperSignalIn vitro proliferation of MCF-7 cells was measured over 5 days with an initial cell density of 3.5 × 103/well and six replicate wells/time point by MTT assay as follows. The medium in each well was replaced with 100 μl of medium containing MTT at 0.5 μg/μl and plates were returned to the incubator for 4 hours. The medium-MTT was then removed and 100 μl of dimethyl sulfoxide added to each well. The plates were kept in agitation for 10 minutes in the dark to dissolve the MTT-formazan crystals. The absorbance of the samples was recorded at 570 nm. The results are presented as the mean ± the standard deviation (SD). The experiment was repeated for three times.MCF-7 cells were washed twice with cold phosphate-buffered saline (PBS) and fixed in cold 70% (v/v) ethanol at 4°C for at least 18 h. Then the cells were harvested and sequentially incubated with RNase A (100 μg/ml) at 37°C for 30 minutes and stained with propidium iodide (PI) (50 μg/ml) in the dark at 4°C for 20 minutes. DNA content was measured by a FACScan cytometer (Becton Dickinson). The experiment was repeated twice.4/100 μl in serum-free DMEM supplemented with 0.1% BSA. The cells were allowed to adhere for 1 hour at 37°C. Nonadherent cells were removed by gentle washing with PBS and adherent cells were evaluated with MTT method and OD570 nm determination. The experiment was conducted in triplicate.For adhesion assay, briefly, triplicate wells were precoated overnight at 4°C with BSA (1% w/v), Matrigel diluted 1:8 in DMEM and fibronectin (10 μg/ml). The cells were seeded at 1 × 10Cells were cultured in DMEM supplemented with 10% FCS on Matrigel (diluted 1:10 in DMEM)-coated 96-well plates for 24 hours. The injury lines were made with a tip on the cells grown at >90% confluency. After being rinsed with serum free DMEM, cells were allowed to migrate in DMEM supplemented with 1% FCS. The area of cell-free wound was photographed with an inverted microscope  equipped with a digital camera  at indicated time points. The wound healing effect was calculated as the ratio of leading edge to initial wound length. The experiment was performed by duplicate.7 MCF-7 or MCF-7/c-Jun cells suspended in 200 μl of PBS were injected s.c. in the right flank. For experimental lung or liver metastasis assays, mice were injected intravenously with 3 × 106 tumor cells in a volume of 200 μl via the lateral tail vein. Primary tumors, lungs and livers were autopsied 3–4 weeks post injection and tissues with metastases were either photographed for gross morphology or processed for histology as described in the following.Five-week-old female Balb/C athymic nude mice were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Science and housed in specific pathogen-free conditions in Good Laboratory Practice facility. For all studies, the mice were allowed to acclimate at least 3 days after receipt of shipment and caged in groups of 6. Before injection, the cells were washed with PBS, harvested by trypsinization, resuspended and kept on ice until injection. Precise cell counts were obtained from samples of the cell suspensions by using a hemocytometer. 2 × 10Tissues were dissected and fixed in 10% buffered formalin and embedded in paraffin wax. Tissue sections (4 μm) were stained with H & E for morphology. The photographs were taken on a Nikon microscope.P < 0.05 was considered statistically significant.Data were expressed as mean ± SD. For proliferation and adhesion assay, the data were compared by the Mann-Whitney test. For wound migration assay, the comparisons were made by repeated-measures ANOVA as appropriate. In order to investigate the effects of c-Jun on tumorigenesis and metastasis of breast cancer cells, pCDNA3.1/c-Jun was transfected into MCF-7 cells. The transfected MCF-7 cells were selected in levels of Geneticin (G418) up to 800 μg/ml for stable c-Jun expression. Individual neomycin-resistant colonies (MCF-7/c-Jun) from pCDNA3.1/c-Jun transfections were isolated, expanded and analyzed for c-Jun expression. Western blot analysis of expression of c-Jun protein in individual clones of stable transfectants is illustrated in Fig. P < 0.01) compared with mock transfected MCF-7 cells. To determine the effect of c-Jun expression on cell cycle progression, the cells were analyzed by PI staining and flow cytometry. A significant increase in S phase cells (from 10.68% to 28.63%) could be observed in MCF-7 cells overexpressing c-Jun, indicating the involvement of c-Jun in positive regulation of cell cycle .The process of tumor cell invasion involved in tumor cell adhesion to the basement membrane, degradation/proteolysis of the basement membrane and tumor cell migration into secondary sites. Cell movement is generally a continuous and dynamic interplay of adhesions at the leading edge and deadhesions at the rear portion of the cells. A certain degree of cell attachment to ECM substrates is necessary for cell motility. Tumor cell lines that are highly invasive and metastatic exhibit a higher degree of adhesion and motility than their lower metastatic counterparts. To investigate the effect of c-Jun expression on the adhesion and motility of MCF-7 cells, the adhesive ability of MCF-7/c-Jun cells was compared with MCF-7/pCDNA3.1 cells. As shown in Fig. p < 0.01).Next the cell motility and the invasive capacity of MCF-7/c-Jun cells were analyzed in a classical wound migration assay. Cell motility into the Matrigel-coated area was determined from micrographs taken at various time points. Compared with the control cells, the motility MCF-7/c-Jun cells was significantly increased Fig.  and 3C, 7 cells subcutaneously into mice. The tumor growth was observed in 4/6 mice injected with MCF-7/c-Jun cells, but parental MCF-7 cells did not produce tumors. Histologically, the tumors were composed of relatively large cells that differ in size and displayed areas with glandula-like arrangement of tumor cells  declare that they have no competing interests.in vivo studies. LY, LQ, YS evaluated the immunohistochemical data. GY, HZ, MH participated in the proliferation, adhesion and motility assays. MY performed flow cytometry analysis. NG participated in the design of the study, supervised the laboratory work, and drafted the manuscript. All authors read and approved the final manuscript.YZ constructed the expression plasmid, carried out immunohistochemical study, performed statistical analysis and participated in writing the manuscript. XP, BF participated in the design of the study and coordinated the study. MS participated in The pre-publication history for this paper can be accessed here:


Yersinia pestis, the causative agent of plague, is a pathogen with a tremendous ability to cause harm and panic in populations. Due to the severity of plague and its potential for use as a bioweapon, better preventatives and therapeutics for plague are desirable. Subunit vaccines directed against the F1 capsular antigen and the V antigen  of Y. pestis are under development. However, these new vaccine formulations have some possible limitations. The F1 antigen is not required for full virulence of Y. pestis and LcrV has a demonstrated immunosuppressive effect. These limitations could damper the ability of F1/LcrV based vaccines to protect against F1-minus Y. pestis strains and could lead to a high rate of undesired side effects in vaccinated populations. For these reasons, the use of other antigens in a plague vaccine formulation may be advantageous.Y. pestis. Additionally, evidence is presented that suggests antibody to YscF is likely not protective by blocking the activity of the TTSS.Desired features in vaccine candidates would be antigens that are conserved, essential for virulence and accessible to circulating antibody. Several of the proteins required for the construction or function of the type III secretion system (TTSS) complex could be ideal contenders to meet the desired features of a vaccine candidate. Accordingly, the TTSS needle complex protein, YscF, was selected to investigate its potential as a protective antigen. In this study we describe the overexpression, purification and use of YscF as a protective antigen. YscF immunization triggers a robust antibody response to YscF and that antibody response is able to afford significant protection to immunized mice following challenge with Yersinia pestis type III secretion complex, as a protective antigen against experimental plague infection. Immunization of mice with YscF resulted in a high anti-YscF titer and provided protection against i.v. challenge with Y. pestis. This is the first report to our knowledge utilizing a conserved protein from the type III secretion complex of a gram-negative pathogen as a candidate for vaccine development.In this study we investigated YscF, a surface-expressed protein of the  Yersinia pestis, the causative agent of plague, causes rapidly progressing disease in humans with a high mortality rate, especially in the pneumonic form of the disease. Due to the severe nature of plague, its ability for aerosol transmission, and the potential for human to human transmission plague is considered to be a disease of high concern as an agent of biological warfare or biological terrorism [Y. pestis, as F1-negative strains have the same LD50 value as F1-positive strains [Yersinia species other than Y. pestis, that could theoretically limit the usefulness of an LcrV based vaccine. While the recombinant subunit vaccines are very effective in experimental animals and offer protection against F1 minus strains of Y. pestis [Y. pestis strains, or the new antigens could be developed as separate vaccine candidates.errorism . For thierrorism . The beserrorism  separateerrorism ,5. Theseerrorism  in mouse strains -9. A sec strains -13. Addi. pestis , the incY. pestis is a conserved virulence mechanism that is absolutely required for virulence of Y. pestis [Y. enterocolitica deficient in production of the translocators  do not translocate Yops into macrophages, demonstrating that the YscF-needle is not sufficient for translocation [The type III secretion apparatus encoded on the low-calcium response (LCR) virulence plasmid, pCD1 in strain KIM , of Y. p. pestis . YscF is. pestis -19. One . pestis . Other r. pestis . This su. pestis -28. AddiSalmonella and Shigella, the YscF homologs  have been demonstrated to form a needle structure that protrudes from the surface of bacterial cells [E. coli (EPEC). EscF is required for "attaching and effacing" (A/E) lesion formation on the intestinal mucosa and for type III secretion of effector proteins [Most currently described pathogenesis-related type III secretion systems possess homologs to YscF. In pathogenic al cells -31. The proteins -34. EscFproteins -34 Howevproteins .Y. pestis and is required for virulence, we sought to determine if YscF could serve as a protective antigen against experimental plague infection. Immunization of mice with His-tagged YscF resulted in a high anti-YscF titer and significant protection against i.v. challenge with Y. pestis. The findings of this study suggest that YscF may be a potential plague vaccine candidate that could be used in conjunction with other plague antigens, or possibly alone if its efficacy can be improved by alternative delivery methods.Based on the fact that YscF is thought to be a surface-expressed protein in yscF was cloned into the overexpression plasmid pET24b (Novagen) to yield a hexahistidine-tag on the C-terminus of YscF (HT-YscF). E. coli BL21(DE3) harboring the HT-YscF expression plasmid, pJM119, was grown in one liter of LB broth [550 reached ~1.0. Cells were harvested by centrifugation and disintegrated by passage through a French pressure cell at 20,000 lb/in2. Subsequent to disintegration, the extracts were clarified by centrifugation at 3,200 × g for 20 min at 4°C. Affinity purification of HT-YscF was performed using Talon resin (BD Clontech) using standard methods. Purity of the recovered protein (> 95 %) was estimated by SDS-PAGE on a 15% (wt/vol) gel followed by staining with a Coomassie blue stain. The purified HT-YscF ran as multiple bands on the gel, regardless of the presence or absence of a reducing agent, such as dithiothreitol (data not shown). A band that corresponded to the predicted size of HT-YscF was the dominant species and other larger bands could also be visualized .To facilitate the purification of YscF, LB broth  containid Figure . Based od Figure  demonstrd Figure  to demonY. pestis, Y. pestis protein extracts from the parental strain KIM8.3002  and extracts from a isogenic yscF deletion strain  were immunoblotted and probed with pooled antiserum from HT-YscF immunized mice  restored YscF reactivity to the HT-YscF serum in the ΔyscF strain  suggesting that the reactivity seen is not induced by immunization with HT-YscF. The higher molecular weight band seen in the culture supernatant fractions could represent a multimer of YscF (possibly a trimer) or YscF in complex with another secreted protein. This higher molecular weight species is likely related to YscF as a similar band is not seen with pre-immune sera (data not shown) or in the ΔyscF strain  for primary immunization and incomplete Freund's adjuvant (IFA) for booster immunizations or with a phosphate-buffered saline  in PBS (grown at 26°C). The mice were observed for 19 days after challenge, and the average doses required to kill 50% of the mice (LD50) for the treatment groups were calculated using the extrapolation method of Reed and Muench [50 value as compared to PBS-immunized mice  controld Muench . Mice th. pestis . The proY. pestis challenge. Due to the strong antibody response, isotyping analysis was performed to determine the predominant isotypes of antibodies produced by mice in response to vaccination with HT-YscF in CFA/IFA. Anti-YscF antibody titers were determined two weeks following the last immunization, prior to challenge. The YscF-specific antibody titers of PBS-immunized mice were below the ELISA assay baseline of 1:800  or in the presence of anti-PcrG . The wt strain was capable of translocating YopE-Elk in presence of both anti-sera, demonstrating that anti-YscF was not capable of blocking Yops translocation and expectedly the ΔyopD strain was still defective for translocation. The experiment likely contained sufficient antibody against YscF to block translocation. In a previous report Pettersson et al used as low as a 1:100 dilution of an anti-LcrV anti-sera with a titer of 1:20,000 for LcrV and in that experiment translocation of Yops into HeLa cells was blocked [Y. pestis into HeLa cells [However, anti-LcrV was able to block Yops translocation by La cells . Since YLa cells . Elk, a La cells ,44. ThisE129-Elk . HeLa ce002  and an , ΔyopD; ) both co blocked . These rLa cells  but coulLa cells ,42, the Y. pestis. This is the first report to our knowledge that has utilized a conserved protein from the type III secretion complex of a gram-negative pathogen as a candidate for vaccine development. This result suggests that type III secretion complexes of other gram negative pathogens could also serve as vaccine targets. YscF and its homologs are obvious targets for use as vaccine candidates as they are surface exposed and are required for virulence in all the systems examined. The protective antibody response elicited by HT-YscF is evidence that YscF is not only expressed during the course of a plague infection, but is also in a location accessible to antibodies at some point in the infectious process. The mechanism of protection by the YscF antibody response is currently under investigation. Essentially three possibilities exist to explain the antibody's protective activity: increased opsonization of the bacteria, enhanced complement binding to the bacterial surface, or direct blocking of Yops translocation into the host cells. Cytotoxicity assays (data not shown) and the ability to translocate Elk-tagged YopE into HeLa cells . The primers used to amplify yscF were HT-YscF Start (5' CGG GAT CCG ATG AGT AAC TTC TCT GGA TTT 3') and HT-YscF Stop (5' CCG CTC GAG TGG GAA CTT CTG TAG GAT GCC 3'). E. coli BL21(DE3) (Novagen) harboring pJM119 was used for HT-YscF overpexpression according to the manufacturer's suggestions. HT-YscF was purified using Talon resin  according the manufacturer's directions.Plasmid pJM119 was constructed by cloning a Y. pestis via the retro-orbital sinus using blunt-feeding needles. Y. pestis used to infect mice was grown overnight in HIB broth, sub-cultured to an A620 of 0.2 absorbance units and grown with shaking to an A620 of 1.0 absorbance unit. Y. pestis cells for infection were harvested by centrifugation and resuspended in PBS. Plate counts were performed to verify CFUs for the infectious doses. Infected animals were monitored for death for up to 19 days, after which survivors were euthanized by CO2 inhalation, according to the guidelines of the Panel on Euthanasia of the American Veterinary Medical Association. All animal work for this project was reviewed and approved by UND's IACUC.For primary immunization 6-to 8-week-old female Swiss-Webster mice were immunized i.p. with 40 μg/mouse His-tagged YscF or phosphate-buffered saline  PBS , and KIM8.3002.2 ΔyopD [Y. pestis strains were grown in heart infusion broth or on tryptose blood agar base medium  at 26°C for genetic manipulations. For physiological studies, growth of Y. pestis was conducted in a defined medium, TMH, as previously described [Bacterial strains used were KIM8-3002 , ΔyscF a.2 ΔyopD . Y. pestescribed . Bacteriescribed . Brieflyescribed ). Bacterescribed .4-Elk . Hexahistidine tagged YscF was visualized using a penta-histidine specific antibody  Alkaline phosphatase-conjugated secondary antibody  was used to visualize proteins by development with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate .Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), using 12.5 % or 15 % (wt/vol) polyacrylamide gels according to the method of Laemmli . Samples2PO4, ph 9.0) at room temperature for 2 h (or overnight at 4°C). The wells were blocked with 200 μl/well blocking buffer  were coated with 100 μl of HT-YscF solution  of 5 to 10) directly to prewarmed medium in the wells of the six-well plates. Plates were then centrifuged at 200 × g at RT for 5 min to achieve contact between the bacteria and the target cells and incubated at 37°C for 4 h.Infection of eukaryotic cells was performed as described previously . Prior t129-Elk was performed as described [Y. pestis strains carrying plasmid pYopE129-Elk were used to infect HeLa cells. After 4 h, the culture supernatants were removed, and the infected adherent cells were lysed by the addition of 100 μl of 2X SDS-PAGE lysis buffer containing Pefabloc  and phosphatase inhibitor (P-2850) cocktail . Samples were boiled for 5 min and loaded onto 12.5 % SDS-PAGE gels, immunoblotted to PVDF membranes and probed with Elk-1 (#9182) or phosphospecific Elk-1 (#9181) antibody preparations . Anti-sera specific for HT-YscF  or the Pseudomonas aeruginosa LcrG homolog, PcrG , were used at dilutions of 1:10 or 1:25 in the infection medium to assess the ability of α-YscF to effect YopE translocation.Detection of Elk-tagged YopE from pYopEescribed . Y. pestyscF for overexpression, purified HT-YscF, characterized the reactivity of antiserum to YscF, assisted with immunization and infection of mice, assisted with the LD50 calculation, and wrote the draft of the manuscript. K. A. D performed immunizations and infections of the mice and performed the antibody isotyping. D. S. B. helped to design the immunization protocol and edited the manuscript. M. L N. conceived of the study, supervised the work, calculated the LD50 and edited the manuscript.J .S. M. cloned


Plasmodium chabaudi chabaudi. In a previous such analysis using the progeny of a genetic cross between P. c. chabaudi lines AS-pyr1 and CB, a location on P. c. chabaudi chromosome 8 containing the gene for merozoite surface protein-1, a known candidate antigen for Strain Specific Protective Immunity, was strongly selected. P. c. chabaudi apical membrane antigen-1, another candidate for Strain Specific Protective Immunity, could not have been evaluated in this cross as AS-pyr1 and CB are identical within the cell surface domain of this protein. Here we use Linkage Group Selection analysis of Strain Specific Protective Immunity in a cross between P. c. chabaudi lines CB-pyr10 and AJ, in which merozoite surface protein-1 and apical membrane antigen-1 are both genetically distinct. In this analysis strain specific immune selection acted strongly on the region of P. c. chabaudi chromosome 8 encoding merozoite surface protein-1 and, less strongly, on the P. c. chabaudi chromosome 9 region encoding apical membrane antigen-1. The evidence from these two independent studies indicates that Strain Specific Protective Immunity in P. c. chabaudi in mice is mainly determined by a narrow region of the P. c. chabaudi genome containing the gene for the P. c. chabaudi merozoite surface protein-1 protein. Other regions, including that containing the gene for P. c. chabaudi apical membrane antigen-1, may be more weakly associated with Strain Specific Protective Immunity in these parasites.Protective immunity against blood infections of malaria is partly specific to the genotype, or strain, of the parasites. The target antigens of Strain Specific Protective Immunity are expected, therefore, to be antigenically and genetically distinct in different lines of parasite. Here we describe the use of a genetic approach, Linkage Group Selection, to locate the target(s) of Strain Specific Protective Immunity in the rodent malaria parasite  In regions of the world where malaria is endemic, anti-parasitic protective immunity against infection with the pathogenic blood stages of the parasites is acquired gradually after repeated exposure to malaria Plasmodium falciparum malaria in humans Plasmodium chabaudi malaria in rodents Natural populations of human malaria parasites P. chabaudi chabaudi, we located a region of the genome on P. c. chabaudi chromosome 8 which contains the gene for MSP-1 P. c. chabaudi strains, AS-pyr1 and CB, used in that study are genetically identical for the cell-surface, ectodomain region of the gene for AMA-1 , the other prominent candidate for SSPI in rodent malaria P. c. chabaudi strains, AJ and CB-pyr10, which are genetically different for the genes for both MSP-1 and AMA-1 . The present analysis has again identified the region on P. c. chabaudi chromosome 8 containing the gene for MSP-1 to be under strong SSPI selection. A weaker SSPI response was also found in the region on P. c. chabaudi chromosome 9 containing the gene for AMA-1. However, no region in the genome of P. c. chabaudi was identified under strength of SSPI selection comparable to that on the region containing the gene for MSP-1. The combined results from these two studies indicate, therefore, that the main force of SSPI is determined by the single, narrow region of the P. c. chabaudi genome containing the gene for the protein antigen MSP-1.In a previous study using Linkage Group Selection (LGS) P. c. chabaudi was induced in groups of female CBA mice, five to six weeks old on first infection, by two successive single strain blood stage-induced infections of either P. c. chabaudi CB or AJ, drug cured with mefloquine of, as described in Materials and Methods. Sixteen weeks after the last mefloquine dose, two CB-immunised mice and two AJ-immunised mice were challenged with a mixture of an equal proportion of blood stage parasites of CB and AJ. A non-immune mouse, which was a batch mate of the immunised mice, was inoculated with the same mixture of blood stage parasites of CB and AJ. Total parasitaemias were measured daily by microscopic analysis of thin blood smears  analysis of DNA samples obtained from the mixed strain infections between 5 and 8 days after inoculation  by strain specific RTQ-PCR see . Single P. c. chabaudi CB and AJ was generated  into groups of four non-immune female CBA mice on days 11 and 12 of infection, respectively. The cross progeny from the non-immune mouse was similarly expanded by subinoculation on day 9 of infection. These parasites were prepared for extraction of parasite genomic DNA for the subsequent analysis, as described in Materials and Methods.A genetic cross between ated see , as descgeny see . The bloP. c. chabaudi were obtained. Of these bands, 197 were present only in AJ and 153 were present only in CB. These were used as AFLP markers for the strains AJ and CB, respectively. The Comparative Intensity (CI) (see The genomic DNA of parasites derived from the CB- and AJ-immune selected cross progeny and from the cross progeny grown in the control non-immunised batch mate was typed with genome-wide quantitative genetic markers produced by Amplified Fragment Length Polymorphism (AFLP) Materials and Methods. 19 contained a single sequence each, all of which were identified in the P. c. chabaudi genome database, at the website http://www.sanger.ac.uk/cgi-bin/blast/submitblast/p_chabaudi. The predicted orthologous loci of these P. c. chabaudi contigs were, thereafter, located within the P. falciparum genome. Because of the high level of conserved synteny between the genomes of P. falciparum and the rodent malaria parasites P. falciparum orthologues of AJ markers could, in turn, be mapped to the equivalent predicted chromosomal positions in the P. c. chabaudi genome. The remaining 5 AJ markers under SSPI selection gave unreadable sequence data. Their physical locations in the P. c. chabaudi genome could not be determined  (12.2% of AJ specific markers) had CIs of less than 50% under AJ-specific immune selection (i.e. intensity reduction of greater than 50% relative to growth of the cross progeny in the non-immune batch mate), listed in termined .P. falciparum genome, one mapped to a region on P. falciparum chromosome 4 which is syntenic with a location on P. c. chabaudi chromosome 7. Nine other markers were located to a region on P. falciparum chromosome 9 which is syntenic with one on P. c. chabaudi chromosome 8. Three markers were located to a region on P. falciparum chromosome 11 which is syntenic with one on P. c. chabaudi chromosome 9. Five additional markers were located to a region on P. falciparum chromosome 12 which is syntenic with one on chromosome 14 of P. c. chabaudi. A final marker responding to SSPI selection was located to a region on P. falciparum chromosome 14 corresponding to one on P. c. chabaudi chromosome 12  and g12 (three markers).In addition, 92 of the 197 AJ markers were located onto ten e groups . In mostP. c. chabaudi chromosome 8 by both physical and genetic mapping  or genetic distance (P. c. chabaudi) from the AJ allele of the P. c. chabaudi msp-1 locus (msp-1 was reduced to undetectable levels (<1%) as measured by strain specific RTQ-PCR in the cross progeny grown under the AJ-specific immune selection compared to its presence at a proportion of 60.0% in the cross progeny grown in the non-immune mouse  following the AJ-specific immune selection were all located on -1 locus . The prone mouse . Thus, tP. c. chabaudi AMA-1  were already very faint in the unselected (control) cross progeny compared to the intensity of the corresponding AFLP bands in the parental strain CB. The general band intensities of the CB markers were below the limit of detection by AFLP in the CB-immune selected cross progeny msp-1 and ama-1 loci using RTQ-PCR. In DNA from parasites of the CB-immune selected cross progeny the CB allele of msp-1 was undetectable (<1%) by RTQ-PCR, compared to 40.0% in the cross progeny grown in the non-immune mouse. At the ama-1 locus, 1.8% of the DNA of parasites carried the CB allele of ama-1 from cross progeny grown in the CB-immunised mouse, compared to 20.4% in the cross progeny grown in the non-immune mouse.In a reciprocal experiment the uncloned progeny of the cross between Plasmodium chabaudi chabaudi which contains the gene(s) for a target of SSPI against the blood stages of this parasite. We have taken two genetically distinct strains of P. c. chabaudi, CB and AJ, which induce SSPI with respect to each other in laboratory mice, and crossed them to produce recombinant progeny. We then applied SSPI selection to the uncloned recombinant progeny by growing the cross progeny in mice made immune to one or the other of the two strains of the parasite (strain specific immunised mice). As a reference (LGS control), the same uncloned progeny was also grown in a non-immune batch mate of the strain specific immunised mice. The cross progeny grown in the CB- or AJ-immunised mouse, or the non-immune batch mate, was screened for the presence and intensity of quantifiable genome-wide AFLP markers distinguishing CB and AJ.Linkage Group Selection is a molecular and genetic approach that applies a specific selection pressure to the uncloned recombinant progeny of a genetic cross between two genotypes (strains) of the same species of malaria parasites that differ in their phenotypic responses under the specified selection pressure Results. Several AJ markers that were located to a region on P. c. chabaudi chromosome 8 formed the deepest identified selection valley in this analysis  containing a gene for the P. c. chabaudi MSP-1, a principle candidate for a target antigen of SSPI. RTQ-PCR analysis confirmed that the uncloned cross progeny carrying the AJ allele of the msp-1 gene was virtually eliminated after selection in the AJ-immunised mouse. The reciprocal result was obtained after selection of the cross progeny in a CB-immunised mouse. In this case parasites carrying the CB allele of the P. c. chabaudi msp-1 locus were virtually eliminated. These results demonstrate that parasites carrying AJ or CB alleles in the region of the msp-1 locus were under very strong selection in AJ- or CB-immunised mice, respectively. A previous LGS analysis of SSPI in the progeny of a genetic cross between strains AS-pyr1 and CB of P. c. chabaudi also found that the region containing the msp-1 locus was under the strongest detected strain specific protective immune selection for this combination of strains P. c. chabaudi chromosome 8 around the msp-1 locus contains a gene or genes encoding a major target antigen or antigens of SSPI.Following growth of the cross progeny in the AJ-immunised mouse, a small proportion of markers of the immunising strain (AJ markers) was judged to be significantly reduced (CI of <50%) relative to the progeny grown in the non-immune mouse. These markers were located in the genome by either physical or genetic mapping, or both, as described in analysis . Within msp-1, several other AJ markers had CIs of less than 50% after selection in the AJ-immunised mouse  by genetic mapping along chromosome 7, but there was uncertainty as to the location of these markers in the genome by physical mapping . It is, therefore, highly unlikely that evidence of the involvement of AMA-1 in SSPI could have been found in the AS-pyr1 and CB cross, nor was it As already noted we had previously conducted LGS analysis of SSPI with a combination of P. c. chabaudi genome that may be under SSPI selection pressure but have not been detected, as no AFLP markers have been mapped on P. c. chabaudi chromosomes 2, 3, 4 and 14 and only a few on chromosomes 10 and 12  NIMP23, which had been raised against the MSP-1 protein of P. c. chabaudi strain AS, inhibited the growth of blood stages of the homologous strain (AS) P. c. chabaudiP. c. chabaudi MSP-1 P. c. chabaudi are, therefore, consistent with the involvement of MSP-1 in SSPI in this parasite.LGS analyses of SSPI have, however, been conducted on the progeny of two different genetic crosses, one between AJ and CB-pyr10 (in this present study) and the other between AS-pyr1 and CB  P. c. chabaudi chromosome 8 which is under strong SSPI selection and which contains the gene for P. c. chabaudi MSP-1. No other region in the P. c. chabaudi genome was found to contain a similarly strong target of SSPI.In conclusion, SSPI to blood stage malarial infections has been observed in malarias of rodents P. c. chabaudi denoted CB-pyr10 and AJ were used in these experiments. CB-pyr10 is a pyrimethamine resistant cloned line derived through pyrimethamine selection from CB (a pyrimethamine sensitive line), originally cloned from isolate CB Thamnomys rutilans, captured in the same locality in the Central African Republic in 1969 and 1970, respectively Two cloned strains of the rodent malaria species Anopheles stephensi. Mice and mosquitoes were maintained as previously described Females of inbred laboratory mouse strains, CBA/Ca and C57BL/6J, were used, aged 5 to 6 weeks old on first infection. These mouse strains will be referred here to as CBA and C57. Mosquitoes were from a laboratory-bred colony of P. c. chabaudi followed by mefloquine cure with slight modifications from the method previously described 5 pRBC of either strain, CB or AJ, of P. c. chabaudi. Blood stage malarial infections were terminated by four consecutive daily doses of 20 mg/kg mouse body weight of mefloquine in 0.1 ml Dimethyl Sulfoxide, by oral gavage. Mefloquine treatment started before parasitaemias exceeded 50% but after they had passed 20% of infected red blood cells. The blood stage parasites were monitored daily by microscopic examination of thin blood smears stained with Giemsa's stain. Following each mefloquine treatment all mice remained blood smear negative. Twenty-six days after the last day of the first mefloquine treatment, mice were given a second i.p. infection with 5×106 pRBC of the P. c. chabaudi strain homologous to the first immunising one. Infections were grown for 5 days and drug cure treated as before.Immunity was induced in groups of CBA mice by two consecutive rounds of infection with 6 pRBC containing an equal proportion of blood stage parasites, CB and AJ. An equivalent inoculum from the same mixture was inoculated into a non-immune mouse, which was a batch mate of the immunised mice. The resulting infections were examined daily by microscopic analysis. A 20 µl sample of tail blood in physiological citrate saline (0.85% (w/v) sodium chloride, 1.5% (w/v) tri sodium citrate dihydrate, in distilled water, adjusted to pH 7.2 with hydrochloric acid) was collected from each mouse 5–8 days post infection, so as to accurately measure proportions of parasites of each strain (CB and AJ) using strain specific RTQ-PCR for CB and AJ alleles of the msp-1 gene Sixteen weeks after the second mefloquine treatment, two mice previously immunised by blood stage parasite infection with either CB or AJ from each group were randomly chosen to test for the presence of SSPI. Each mouse was challenged with a mixture of 5×106 pRBC per mouse into three groups of C57 mice (four mice each). The parasitaemias from the mixed strain infections were followed microscopically on thin tail blood smears stained with Giemsa's stain. Six days post inoculation of the parasites, when parasitaemias were ∼20% and the presence of gametocytes of both sexes was confirmed microscopically, mice from each mixed strain infection group were anaesthetised and placed on a mosquito cage, each containing ∼250 female mosquitoes. Mosquitoes were aged 5–7 days post emergence from pupae. The mosquitoes were allowed to feed on the mice for 20 minutes without interruption. The mice were thereafter humanely killed before recovery from the anaesthesia. Eight days after the blood meal, samples of mosquitoes from each cage were dissected and examined for the presence of oocysts on their midguts. 11 mosquitoes from those fed on the 1∶1 CB:AJ mixture had a mean of 9.5 oocysts per gut ; 16 mosquitoes from those fed on the 1:2 CB:AJ mixture had a mean of 4.06 oocysts per gut (SEM = 3.96); 18 mosquitoes from those fed on the 2:1 CB:AJ mixture had a mean of 3.13 oocysts per gut (SEM = 6.02).Single strain infections with CB and AJ were individually grown in donor C57 mice. Parasites of the two strains were harvested from the donors and accurately mixed to produce three inocula in proportions of 1∶1, 1∶2 and 2∶1 of CB and AJ, respectively. The mixtures were inoculated i.p. at 10Sixteen days after the blood meal, when sporozoites were present in the mosquito salivary glands, the mosquitoes were allowed to feed on 6 non-immune CBA female mice in order to naturally transmit sporozoites of the progeny of the CB×AJ genetic crosses. All surviving mosquitoes of these three batches were then dissected for sporozoites from the salivary glands . The glands were gently crushed in 0.2–0.4 ml volumes of 1∶1 Foetal Bovine Serum (GIBCO BRL): Ringer's solution  and injected i.p. into 8 non-immune CBA mice. All 14 mice, naturally infected or inoculated with sporozoites dissected from salivary glands of the mosquitoes, became infected. The blood stage parasites representing the progeny of the genetic cross were harvested on day 9 post infection  and pooled prior to inoculation of mice previously immunised with parasites of either one or the other of the parental strains CB or AJ for SSPI selection, or else into a non-immune batch mate. The remaining pooled blood was frozen as stabilates and stored in liquid nitrogen.From the data on the number of oocysts per mosquito and the number of mosquitoes dissected for inoculation of sporozoites (given above), it is possible to estimate the likely maximum numbers of recombinant lines that a genetic cross could have generated. On an assumption that gametes of the two parasite strains are generated in equal numbers in the mixed strain infection used to make a genetic cross, and that fertilisations occur randomly between them, half of all fertilisations will generate hybrid zygotes between the gametes of the two parental strains of parasite, while the second half will derive equally from the self fertilisations representing one or other of the two parental strains. Each hybrid zygote undergoes meiosis and produces four recombinant progeny lines. Thus, on average, for every four oocysts in such a genetic cross, there will be two parental and two hybrid oocysts, yielding a total of eight recombinants. Overall, therefore, there are, on average, two recombinant lines for every oocyst present. Using this logic we were able to estimate the likely maximum number of recombinant lines present in the pooled cross progeny .6 pRBC of the uncloned cross progeny. At time of infection with the cross progeny, the immunised mice were twenty weeks after the last day of the second mefloquine treatment (see above). The blood stage parasites from the cross progeny grown in these mice which had been previously immunised with CB or AJ, were designated “CB-immune selected cross progeny” or “AJ-immune selected cross progeny”, respectively. An equal number of the cross progeny designated “non-immune selected cross progeny” was passaged into a non-immune batch mate of the immunised mice. The resulting infections were followed by microscopic examination of thin blood smears stained with Giemsa's stain. The uncloned cross progeny was grown in the CB- and AJ-immunised mice for 11 and 12 days, respectively. In the non-immune batch mate, the cross progeny was allowed to grow for 9 days. To grow parasites in sufficient quantities to prepare DNA for subsequent analysis, blood was, thereupon, collected, and 106 pRBC from each mouse were inoculated into four non-immune mice. The parasites were grown in these mice to parasitaemias of ∼35–50% before harvesting and preparing them for DNA extraction, as previously described by Grech et al., The blood stage parasites from a total of 14 non-immune CBA mice  were harvested in physiological saline and pooled to produce the blood inoculum containing the uncloned CB×AJ cross progeny. One mouse from the batches immunised with blood stage parasite of either CB or AJ and previously tested for SSPI to these strains was inoculated i.p. with 5×10P. c. chabaudi genome affected by the SSPI selection, we generated 33P-radiolabelled AFLP markers from genomic DNA of the CB- and AJ-immune selected cross progeny, the non-immune selected cross progeny and the two parental cloned strains (CB and AJ) by the method of Grech et al., i) were, thereafter, quantitatively compared to the RII of the same markers derived from the non-immune selected cross (RIIni). These proportions were, then, used to calculate the “Comparative Intensity (CI)”- a measurement of proportional changes of strain specific AFLP markers between the immune and non-immune selected cross progeny. The CI is defined by the RII of a strain specific AFLP marker of the immune-selected cross progeny (RIIi) divided by the RII of the relevant marker of the non-immune selected cross progeny (RIIni), and expressed as a percentage, i.e., CI = (RIIi/RIIni)×100 To locate regions of the Strain specific AFLP markers that had CI reduced below 50% following the strain specific immune selection were identified and characterised by the methods of Hunt et al., P. c. chabaudi cloned strain AS, http://www.sanger.ac.uk/cgi-bin/blast/submitblast/p_chabaudi, were searched using the BLASTN (DNA vs. DNA) option with sequences derived from the AFLP markers. Genomic contigs of P. c. chabaudi which contained DNA sequences corresponding to the relevant AFLP marker with lowest Probability score (a cut-off of 90% sequence identity) were obtained. The chromosomal positions of these contigs have not been mapped physically in the P. c. chabaudi genome. Sequences of whole P. c. chabaudi contigs were, thereafter, used to locate orthologous loci in the Plasmodium falciparum (cloned strain 3D7) genome database http://www.ncbi.nlm.nih.gov/sutils/blast_table.cgi?taxid = Protozoa&database), using the blastn option. The genomic locations of the P. falciparum orthologues were, in turn, used for physical mapping to chromosomal locations in the genome of P. c. chabaudi through the conserved genetic synteny between the human malaria parasite P. falciparum and rodent malaria parasites Eight-fold coverage contigs of the genome of P. c. chabaudi genetic linkage map which was constructed with reference to the progeny of a genetic cross between P. c. chabaudi strains AS and AJ . The msp-1 specific primers used in the assay were: the CB forward and reverse primers: 5′-AGTTGTTCCTGTGGCAG-3′ and 5′-CTGTTACAACCCAAACC-3′; and the AJ forward and reverse primers: 5′-ACTGAAGCAACAACACCAGC-3′ and 5′-GTTGTTGATGCACTTGCGGGTTC-3′. RTQ-PCR reactions for the CB and AJ alleles of msp-1 were set up, as previously described ama-1 specific primers used in the assay were: the CB forward and reverse primer 5′-AGGTTTCATTATTAACACGAG-3′ and 5′-GATTACTTTTGTC ATAAACAGCG-3′; and the AJ forward and reverse primer 5′ CTAAATCATTCTTAGA CCC-3′ and 5′-GGCATAATTTTTATATTCTG-3′. The positions at which nucleotides of the CB and AJ ama-1 alleles mismatched are shown in bold. RTQ-PCR reactions for quantification of the CB and AJ alleles of ama-1 were performed in a 10 µl volume of standard buffer containing 4 µM of forward and reverse primers in 3 and 2.5 mM MgCl2, respectively. The PCR conditions used were as follows: An initial “Hot Start” at 95°C for 600 s followed by 40 cycles of 95°C with a 0 s hold, cooling at 20°C/s to 58°C with a 7 s hold for CB or cooling at 20°C/s to 56°C with a 7 s hold for AJ, reheating at 20°C/s to 72°C with a 15 s hold for CB or 10 s hold for AJ and finally heating at 20°C/s to 72°C with a 0 s hold. Melting curve data for each PCR run was produced, as follows: Heating at 20°C/s to 95°C with 0 s hold, cooling at 20°C/s to 65°C with 30 s hold and reheating at 0.2°C/s to 95°C in a continuous data acquisition mode. The final step of RTQ-PCR was at 20°C/s to 40°C with a 60 s hold. Data obtained from each LightCycler run were checked to ensure that the correct strain specific melting peak and no other non-specific amplicon was produced. The DNA concentrations measured on the LightCycler were converted into relative proportions of the AJ and CB alleles for msp-1 or ama-1 within each sample analysed.RTQ-PCR analysis was performed on a LightCycler instrument (Roche Diagnostics) as described by Cheesman et al.,


The second-generation Innotrac Aio! cTnI assay was found to be superior to the first-generation assay with regard to precision in the low concentration range. The stability of the cTnI level was best in the  serum, lithium-heparin plasma, and lithium-heparin whole blood samples  assay. The Innotrac Aio! second-generation cTnI assay was           compared with the Abbott AxSYM first-generation cTnI, Beckman Access AccuTnI, and Innotrac            Aio! first-generation cTnI assays. We studied serum samples from 15 patients with positive             rheumatoid factor but with no indication of myocardial infarction (MI). Additionally, the stability              of the sample with different matrices and the influence of hemodialysis on the cTnI              concentration were evaluated. Within-assay CVs were 3.2%–10.9%, and              between-assay precision ranged from 4.0% to 17.2% for cTnI. The functional sensitivity              (CV = 20 %) and the concentration giving CV of 10% were approximated to be 0.02 and 0.04, respectively. The assay was found to be linear within the tested range of 0.063–111.6 μ g/L. The correlations between the second-generation Innotrac Aio!, Access, and AxSYM cTnI assays were good (


Granulocyte-colony stimulating factor (G-CSF) is known as a powerful regulator of white blood cell proliferation and differentiation in mammals. We, and others, have shown that G-CSF is effective in treating cerebral ischemia in rodents, both relating to infarct size as well as functional recovery. G-CSF and its receptor are expressed by neurons, and G-CSF regulates apoptosis and neurogenesis, providing a rational basis for its beneficial short- and long-term actions in ischemia. In addition, G-CSF may contribute to re-endothelialisation and arteriogenesis in the vasculature of the ischemic penumbra. In addition to these trophic effects, G-CSF is a potent neuroprotective factor reliably reducing infarct size in different stroke models.Here, we have further delayed treatment and studied effects of G-CSF on infarct volume in the middle cerebral artery occlusion (MCAO) model and functional outcome in the cortical photothrombotic model. In the MCAO model, we applied a single dose of 60 μg/kg bodyweight G-CSF in rats 4 h after onset of ischemia. Infarct volume was determined 24 h after onset of ischemia. In the rat photothrombotic model, we applied 10 μg/kg bodyweight G-CSF daily for a period of 10 days starting either 24 or 72 h after induction of ischemia. G-CSF both decreased acute infarct volume in the MCAO model, and improved recovery in the photothrombotic model at delayed timepoints.These data further strengthen G-CSF's profile as a unique candidate stroke drug, and provide an experimental basis for application of G-CSF in the post-stroke recovery phase. Granulocyte-colony stimulating factor (G-CSF) is a cytokine that stimulates generation of neutrophilic granulocytes, and has been in clinical use for more than 10 years in indications related to counteracting neutropenia, or for bone-marrow transplantations . RecentlCharacterised by this multimodal array, G-CSF appears beneficial for short-term damage parameters such as infarct size, and long-term effects on functional outcome . Based Here we further explore the window of opportunity for G-CSF treatment in firstly, the most accepted model for evaluating neuroprotective effects, rat middle cerebral artery occlusion (MCAO), and secondly, the cortical photothrombotic stroke model for detecting effects on long-term functional outcome.We recently reported an infarct-reducing capacity of G-CSF after middle cerebral artery occlusion (MCAO) when treatment was initiated at 30 min and 2 h after onset of ischemia ,6. Here 3 (n = 24) vs. 223.3 ± 27.3 mm3 (n = 18), p < 0.05; Figure 3 vs. 144.3 ± 22.7 mm3; p < 0.05) as well as the subcortical  part of the infarction.We observed a robust infarct volume reduction by 33 % in the total infarct volume as compared to vehicle-treated rats  did not differ between the groups. In addition, there were no differences in mortality or body weight between all groups (not shown).Sensorimotor deficits in the rotarod test were obvious in vehicle-treated ischemic animals compared to sham-operated rats in the rotarod for up to 6 weeks after the insult Figure . G-CSF tHere we demonstrate a robust infarct-reducing effect of a 4 h treatment delay with G-CSF in a severe hemispheric stroke model (MCAO), viewed as the gold standard for testing neuroprotective effects. The end point for determining infarct volume was 24 h following onset of ischemia, a standard timepoint for neuroprotective studies. We know, however, that the effect of G-CSF is not transient as has been discussed with some experimental drugs, but is also detectable when examining the brain at 72 h post ischemia after a combined CCA/distal MCA occlusion . The achSupport for a delayed window of opportunity for G-CSF in acute stroke models comes from three other independent studies. Treatment effects were investigated in comparatively mild ischemia models producing small hemispheric or cortical infarctions appropriate to assess recovery effects rather than to test the neuroprotective potential of a drug in the acute phase. Six et al. have shown a stunning 55% infarct reducing effect when treatment was delayed for 24 h after transient MCAO in the mouse with a relatively small sample size of n = 6 per group . Shyu et3 to 72.5 mm3 in the vehicle treated group, Figure By comparing the differences of total infarct volumes without and with edema correction, our data indicate an absolute reduction in edema volume in favor for G-CSF  our datIn addition to a rational mode-of-action within stroke pathophysiology, any candidate for further stroke drug development should be explored according to the STAIR criteria . G-CSF fThe effect on functional outcome appears as a separable activity of G-CSF, independent of its neuroprotective potential. This makes G-CSF a highly attractive candidate for recovery enhancement in the subacute or chronic phase of a stroke and increases the likelihood for detecting clinical outcome improvement in acute stroke trials.Aiming at the two principal modes-of-action of G-CSF in the brain, antiapoptosis and regeneration enhancement, we have both studied effects on infarct size with delayed treatment in a severe MCAO model, as well as functional sensorimotor outcome in the cortical photothrombotic model. We find that G-CSF has an acute time window of at least 4 h post-stroke, and an astonishing time window for effects on functional outcome of three days after stroke. The strong effects on functional outcome distuinguish G-CSF from most other candidate stroke drugs in development.Our findings further strengthen confidence in the efficacy of G-CSF both for infarct size reduction, and improvement of functional parameters, and are important guiding lines for future clinical development.2O, 30% O2, and 1% halothane. The right femoral vein was cannulated and used for drug delivery. During the experiment, core body temperature was monitored and maintained at 37°C by a thermostatically controlled heating pad . Middle cerebral artery occlusion (MCAO) was induced with a silicon-coated  4-0 nylon filament (Ethicon) that was introduced into the common carotid artery and advanced into the internal carotid artery as described previously [Male Wistar rats weighing 280 to 320 g received inhalation anesthesia with 70%Neviously . SuccessInfarct volumes were determined 24 h after induction of ischemia by TTC staining as described previously . 2 mm seMale Wistar rats were anesthetised with an intramuscular injection of 100 mg/kg bodyweight ketaminehydrochloride (Ketanest) and 8 mg/kg bodyweight xylazine hydrochloride (Rompun). Anesthesia was maintained with 50 mg/kg bodyweight ketaminehydrochloride and 4 mg/kg bodyweight xylazine hydrochlorideif necessary. A PE-50 polyethylene tube was inserted into the right femoral artery for continuous monitoring of mean arterial blood pressure, and blood gases. The right femoral vein was cannulated by a PE-50 tube for treatment infusion. During the experiment rectal temperature was monitored and maintained at 37°C by a thermostatically controlled heating pad . Photothrombotic ischemia was induced in the rat parietal cortex according to the method of Watson et al. . AnimalsIn all animals Rotarod testing was performed during the light cycle before ischemia after a training period of 3 days, and at 1,2,3,4,5 and 6 weeks after ischemia by an investigator blinded to the experimental groups. Rats were placed on an accelerating Rotarod cylinder (increased speed from 4 to 40 rpm within 5 min). The time the animals remained on the Rotarod was measured, and the trial ended if the animal fell off the rungs or gripped the device and spun around for two consecutive revolutions without attempting to walk on the rungs. An arbitrary time limit of 500 sec was set for the rats on the Rotarod cylinder in training and in the testing procedures. The animals were trained 3 days before ischemia, and the mean duration (sec) on the device was recorded with three measurements.All animal experiments were performed in accordance with national and international regulations, and were approved by the appropriate government authorities. All experiments were performed in a fully randomised and blinded fashion.The values presented in this study are means ± SEM. The two-tailed t-test was used to determine significant difference of infarct volumes. Behavioural measurements were analysed using ANOVA and the Student-Newman-Keuls post hoc test. Area under the curve (AUC) was calculated using the trapezoidal algorithm. Linear regressions were compared using coincidence testing. Statistics were performed with NCSS 2004  or Primer of Biostatistics software. A p value <0.05 was considered statistically significant.RW, CP, CK, RL carried out the animal studies, and participated in the data analysis. AS and WRS carried out data analyses. AS, WRS, SS, AB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Opioid antagonism was provided by three or four doses of naltrexone 50 mg, given at 12-hour intervals pre- and post-OROS® hydromorphone dosing. Plasma samples for pharmacokinetic analysis were collected pre-dose and at regular intervals up to 48 hours post-dose (72 hours for the 64-mg dose), and were assayed for hydromorphone concentration to determine peak plasma concentration (Cmax), time at which peak plasma concentration was observed (Tmax), terminal half-life (t1/2), and area under the concentration-time curve for zero to time t (AUC0-t) and zero to infinity (AUC0–∞). An analysis of variance (ANOVA) model on untransformed and dose-normalized data for AUC0-t, AUC0–∞, and Cmax was used to establish dose linearity and proportionality.In an open-label, four-way, crossover study, 32 healthy volunteers were randomized to receive a single dose of OROSmax (12.0–16.0 hours) and mean t1/2 (10.6–11.0 hours) were found to be independent of dose. Regression analyses of Cmax, AUC0–48, and AUC0–∞ by dose indicated that the relationship was linear  and that the intercept did not differ significantly from zero (P > 0.05). Similar analyses with dose-normalized parameters also indicated that the slope did not differ significantly from zero (P > 0.05).The study was completed by 31 of 32 subjects. Median T® hydromorphone are linear and dose proportional for the 8, 16, 32, and 64 mg doses.The pharmacokinetics of OROSClinical Trials.gov NCT00398957 Hydromorphone hydrochloride (HCl), which is available in immediate- and extended-release formulations, is a semi-synthetic opioid agonist that has been used widely for many years in the treatment of acute and chronic pain. A number of studies have demonstrated the efficacy and tolerability of hydromorphone in comparison with morphine and other opioid analgesic agents . When fo® Push-Pull™ osmotic pump delivery system developed by ALZA Corporation  [® hydromorphone  are achieved after 48 hours  and are maintained throughout the 24-hour dosing interval [® hydromorphone is taken immediately after a high-fat meal [To improve pain relief and provide convenient dosing for patients with severe chronic cancer and non-cancer pain, a novel 24-hour controlled-release formulation of hydromorphone is currently being investigated. This formulation uses the patented OROSlto, CA) -8, and alto, CA) . Moreoveinterval . An initfat meal .® hydromorphone with naltrexone, an opioid antagonist, under fasting conditions resulted in a 39% increase in Cmax, but there was no significant change in Tmax, AUC0-t, or AUC0–∞ [® hydromorphone at daily doses of 8, 16, 32, and 64 mg.Co-administration of OROSr AUC0–∞ . These rStudy volunteers were non-smoking, healthy male and female adults between 19 and 50 years of age. Their body weight was required to be between 61 and 100 kg and within ± 10% of the recommended weight range for height and body frame (1984 Metropolitan Height and Weight Tables). Results of the baseline screen were required to be negative for drugs of abuse . Subjects were required to have no clinically significant deviations from normal in laboratory results. All participants provided written informed consent. The study was approved by the Institutional Review Board and was carried out according to the Declaration of Helsinki and subsequent revisions.Subjects who were intolerant of, or hypersensitive to, opioid agonists or antagonists were excluded, as were those with opioid dependency. Other exclusion criteria included gastrointestinal disorders; compromised cardiac, respiratory, renal, or hepatic function; psychiatric abnormalities; and significant hematologic, metabolic, or central nervous system disorders. Study participation did not permit any subject to take any long-term medication, enzyme-altering agents, recreational drugs, or an investigational agent within 30 days of beginning the study.® hydromorphone for dose proportionality after administration of a single oral dose of 8, 16, 32, and 64 mg.This was an open-label, randomized, four-way crossover study designed to examine the pharmacokinetic profile of once-daily OROSBased on the assumption that the within-subject variability is less than 20%  and that there is a 5% difference between treatments, a sample size of 30 subjects was estimated to provide 80% power to demonstrate equivalence at the 0.05 level of significance.® hydromorphone 8, 16, 32, and 64 mg, given after a 10-hour overnight fast), with a 7-day washout period between treatments. The order in which treatments were received was determined according to the predetermined randomization schedule. Naltrexone 50 mg was administered 12 hours before, with, and 12 hours after OROS® hydromorphone in all groups, with an additional 50-mg dose of naltrexone administered 24 hours after the 64-mg dose of OROS® hydromorphone. Naltrexone was administered to minimize adverse events following the higher doses of OROS® hydromorphone in these opioid-naïve subjects, and was given concomitantly with each dose level of OROS® hydromorphone to facilitate dose-proportionality comparisons.Subjects received each of the four treatments  and at 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, 36, 42, and 48 hours post-dose. Additional samples were taken at 56, 64 and 72 hours after the 64-mg dose. Plasma hydromorphone concentrations were measured using a validated LC/MS/MS method  covering a range of 0.05 to 10 ng/mL. Calibration standards prepared for each of the sample sets were used to calculate the inter-day precision of the assay. The coefficients of variation for the standards ranged from 1.7% to 9.9%. The absolute deviations ranged from 0.05% to 2.6%.max), time at which peak plasma concentration was observed (Tmax), terminal half-life (t1/2), and the area under the concentration-time curve from time 0 to time t (AUC0-t) and from time zero to infinity (AUC0–∞). The non-compartmental pharmacokinetic parameters described above were estimated using macros built in Excel .Based on the measured hydromorphone concentration, the following parameters were calculated: peak plasma concentration  regression model to establish dose linearity and dose proportionality. All tests were two-sided at the 0.05 level of significance. Tmax was analyzed non-parametrically, without dose-normalization, using the Wilcoxon matched-pairs test for each pairwise comparison; the 95% confidence interval (CI) for the difference in treatment medians was constructed. Data for t1/2 were summarized using descriptive statistics. The apparent elimination-rate constant (K) for each subject was estimated by linear regression of the log-transformed concentration during the terminal log-linear decline phase of the curve. Terminal half-life was estimated as 0.693/K.Untransformed and log-transformed (ln) data for CThirty-two healthy volunteers were enrolled in the study, 8 in each of four treatments, with at least 24 subjects expected to complete the study. They were primarily male (63%) and Caucasian 81%), with a mean age of 33 years . Analysis of Cmax, AUC0-t, and AUC0–∞ by dose indicated that the relationship was linear (P ≤ 0.05) and that the intercept did not differ significantly from zero . When this subject was excluded, Cmax variability for the 8-mg dose was similar to the other doses. No significant gender-by-treatment interactions were observed .Mean dose-normalized pharmacokinetic parameters for OROSAt least one adverse event was experienced by 21 of the 32 subjects (66%). All events were of mild or moderate intensity, and none were considered serious. Headache, asthenia, and nausea were the most common adverse events, occurring in 31%, 28%, and 28% of patients, respectively, during one or more of the treatment periods. The adverse events for each dose group are shown in Table ® hydromorphone. The time to achieve maximum plasma concentration was independent of dose. Near-maximum plasma concentrations were reached approximately 6 hours after dosing, and plasma concentrations were maintained at or near maximum levels throughout a 30-hour period, consistent with once-daily dosing. Beginning 24 to 30 hours post-dose, plasma hydromorphone concentrations declined slowly, with an apparent terminal half-life of approximately 10 hours. This is longer than the half-life of immediate-release hydromorphone (2–3 hours), which has been determined from studies with intravenous formulations [® hydromorphone [® hydromorphone.The results of this study indicate that plasma hydromorphone concentrations and overall exposure to hydromorphone are proportional to the administered dose (over the 8- to 64-mg dose range) with OROSmorphone . The obs® hydromorphone for the dose range studied. Although limited, these data do suggest that there are no clinically relevant differences between males and females with respect to the pharmacokinetics of OROS® hydromorphone.An exploratory analysis suggested no influence of gender on the pharmacokinetics of OROS® hydromorphone doses, indicating no dose relationship with the incidence of adverse events. Adverse events were consistent with those expected for an opioid agonist and antagonist and primarily affected the digestive and central nervous systems. No serious adverse events were reported during the study.Safety results were consistent for all four OROS® hydromorphone and its pharmacokinetic parameters were found to be proportional to the orally administered dose over the dose range studied (8 mg to 64 mg). Plasma concentrations achieved the maximal level by approximately 16 hours after single administration, independently of dose, and remained near that level for up to 30 hours. Adverse events were consistent with those expected for an opioid agonist and antagonist.Plasma concentrations of OROSThe authors declare that they have no competing interests.Financial Competing InterestsIn the past five years none of the authors of this manuscript have received reimbursements, fees, funding, or salary from an organization that may in any way gain or lose financially from publication of this manuscript, either now or in the future.The authors of this manuscript do hold stock in ALZA Corporation but the stock price is not affected by the product alone or the publication.There are no patents filed based on the data disclosed in this publication. The authors have not received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript.The authors do not have any other financial competing interests.The authors do not have any non-financial competing interests  to declare in relation to this manuscript.GS reviewed the pharmacokinetic and statistical results and authored the related sections. EX performed all the pharmacokinetic and statistical analysis. SKG participated in the conception of OROS hydromorphone product and contributed to the design of the study. JT reviewed the safety results and authored the related sections. All authors read and approved the final manuscript. All authors wish to acknowledge Philip Sjostedt and PharmaGenesis, Inc., in preparation of this manuscript.The pre-publication history for this paper can be accessed here:


Exhaled breath condensate (EBC) is a non-invasive method to assess airway inflammation and oxidative stress and may be useful in the assessment of childhood asthma.Exhaled 8-isoprostane, a stable marker of oxidative stress, was measured in EBC, in children (5–17 years) with asthma  and 11 healthy control.Mean exhaled 8-isoprostane concentration was significantly elevated in steroid-naïve asthmatic children compared to healthy children 9.3 (SEM 1.7) vs. 3.8 (0.6) pg/ml, p < 0.01. Children on inhaled steroids also had significantly higher 8-isoprostane levels than those of normal subjects 6.7 (0.7) vs. 3.8 (0.6) pg/ml, p < 0.01. Steroid-naïve asthmatics had higher exhaled nitric oxide (eNO) than those of controls 28.5 (4.7) vs. 12.6 (1.5) ppb, p < 0.01. eNO in steroid-treated asthmatics was similar to control subjects 27.5(8.8) vs. 12.6(1.5) ppb. Exhaled 8-isoprostane did not correlate with duration of asthma, dose of inhaled steroids or eNO.We conclude that 8-isoprostane is elevated in asthmatic children, indicating increased oxidative stress, and that this does not appear to be normalized by inhaled steroid therapy. This suggests that 8-isoprostane is a useful non-invasive measurement of oxidative stress in children and that antioxidant therapy may be useful in the future. Anti-inflammatory drugs such as inhaled corticosteroids now are the mainstay of treatment in childhood asthma but measurement of airway inflammation using traditional invasive procedures is not feasible in children. Bronchial biopsy is invasive and non-invasive tests such as spirometry do not represent the true state of inflammation . Less inExhaled breath condensate analysis is simple to perform, is effort-independent, non-invasive and rapid ,3. VarioIncreased oxidative stress is a feature of airway inflammation in asthma and inflammatory cells, such as eosinophils, neutrophils, macrophages, and mast cells all produce reactive oxygen radicals . 8-isoprNormal and asthmatic children 2–18 years of age who were able to co-operate with the measurements were enrolled into the study. The diagnosis of asthma was based on a history of repetitive cough, breathlessness and wheeze responsive to bronchodilators with or without inhaled steroid ). Exhaled NO [Children on inhaled corticosteroids (n = 12) were divided into two groups; those on ≤ 600 μg/day  and those on >600 μg/day . Atopic status was assessed by skin prick test to 4 common allergens  at an expired flow of 50 ml/second. This equipment has a sensitivity of ± 1.5 ppb and a precision of ± 2.5 ppb. A direct digital reading is obtained and average of two readings was taken in each child. As spirometric maneuvers are known to affect NO readings, exhaled NO measurement was done prior to lung function testing.1 % predicted was calculated for each child.The airway function determination was performed by dry spirometer . The highest of three consecutive measurements was taken. FEVSubjects breathed tidally for 10 minutes into the condenser , wearing a nose clip. Exhaled condensate was frozen at -20°C elsius, after defrosting the sample was aliquoted into small plastic tubes and stored at -80°C for later analysis. This has been shown not to affect 8-isoprostane concentrations over six months of storage [8-isoprostane was assayed by an enzyme-linked immunosorbent assay . The lowest detection limit of the assay was 4.5 pg/ml and the intra and interassay correlation coefficient was ≤ 10% .All data are expressed as means ± SEM. Comparison of demographic data was done by Chi-square test. Continuous data of two subgroups were tested for significant difference by unpaired Student's t-test for normally distributed data. Correlations were performed by Pearson's test for normally distributed data, and by Spearman's test for non-Gaussian data. Significance was considered when p < 0.05. Reproducibility was assessed by Bland-Altman plot of the paired values of exhaled 8-isoprostane of the asthmatic children ,11.Aspergillus fumigatus). The mean eNO was significantly raised in steroid-naïve asthmatic children (28.5 ± 4.7 ppb) compared to normal subjects , but was not significantly increased in the steroid-treated group compared to the healthy controls . There was a wide scatter of eNO levels in the steroid-treated group.We studied 11 normal control subjects, 13 steroid-naïve and 12 steroid-treated asthmatic children. There was no significant difference in age and sex of children between these three groups . The asthmatic children using inhaled steroids had a trend towards lower 8-isoprostane levels than that of steroid-naïve group ; their results were significantly higher than in control subjects  .There was no correlation between 8-isoprostane concentrations and exhaled NO Fig.  or FEV1%The second group of asthmatic children on whom reproducibility of the measurements was analyzed comprised of 10 inhaled steroid-treated asthmatics . The doses of inhaled steroids in these children ranged from 200 to 2000 μg/day. We found that the Bland-Altman plot of repeat values of 8-isoprostane performed 10 minutes apart in these 10 asthmatic children demonstrated good reproducibility. The intra-class correlation coefficient (ICC) of the two readings was 0.98 and the correlation coefficient (r) was 0.98 .We have shown that 8-isoprostane is detectable in exhaled breath condensate of children, with significantly higher concentrations in exhaled breath of asthmatic children compared to healthy controls. Steroid-treated asthmatic patients had a trend towards lower levels of 8-isoprostane in exhaled breath, although the concentrations were still significantly higher than in normal subjects. When the exhaled breath condensate was assessed for reproducibility of 8-isoprostane it was found to be highly reproducible (ICC = 0.98).in vivo, since it is a stable product of oxidative metabolism. Our study revealed that asthmatic children had significantly higher levels of exhaled 8-isoprostane than those in healthy volunteers. 8-isoprostane is predominantly formed by oxidative metabolism of arachidonic acid via a non-enzymatic reaction, but a small amount of 8-isoprostane may be formed by a cyclooxygenase pathway [1% predicted. This temporal measurement would enable us to determine the utility of exhaled 8-isoprostane in predicting an attack of asthma. In the steroid-naïve group most patients were mild and we did not have enough patients with severe disease to study whether exhaled 8-isoprostane levels reflects disease severity as in adults [We studied 36 children, including steroid-naïve asthmatics, asthmatic patients on inhaled steroids and normal controls. The children were age and sex-matched and the youngest child who was able to undertake the test was 5 years. Measurement of 8-isoprostane is a useful marker to assess the oxidative stress of asthma  pathway . Measuren adults . We founWe found that eNO was significantly higher in asthmatic children compared to normal controls, but were reduced in steroid-treated patients, in agreement with other studies . Other aOur study shows that elevated 8-isoprostane is detectable in the exhaled breath condensates of children with asthma and may be used as a non-invasive measurement of oxidative stress in childhood asthma. The persistence of high levels of 8-isoprostane in spite of inhaled steroids suggests that other treatments such as anti-oxidants might be beneficial.


Xerocomus chrysenteron (XCL) has been isolated recently. It shows insecticidal activity and has antiproliferative properties.Lectins are carbohydrate-binding proteins which potentially bind to cell surface glycoconjugates. They are found in various organisms including fungi. A lectin from the mushroom d of 5.2 μM for the XCL:N-acetylgalactosamine interaction at 27degreesC. Higher affinities were observed at lower temperatures and higher osmotic pressures. The dissociation constant was five hundred times higher for the disaccharide beta-D-Gal(1–3)-D-GalNAc, Thomsen-Friedenreich (TF) antigen (Kd of 0.94 μM). By using fetuin and asialofetuin in interaction with the XCL, we revealed its ability to recognize the Thomsen-Friedenreich motif on glycoproteins.As the monosaccharide binding specificity is an important determinant of lectin function, we determined the affinity of XCL for the galactose moiety. Isothermal titration calorimetry studies revealed a dissociation constant KThe XCL antiproliferative effect and the TF antigen specificity presented in this work suggest that XCL and ABL may have similar binding mechanisms. The recent structure determination of these two proteins lead us to analyse these interactions in the light of our thermodynamic data. The understanding of this type of interaction may be a useful tool for the regulation of cell proliferation. Lectins are carbohydrate-binding proteins found in various organisms including fungi ,2. DespiXerocomus chrysenteron (XCL) [Agaricus bisporus lectin (ABL), another mushroom lectin well known for its reversible antiproliferative effects [Mushroom lectins have captured the attention of investigators on account of their antiproliferative, immunomodulatory, antitumor and cytotoxic activities, and more than 50 mushroom lectins have been reported [on (XCL) . The X ron (XCL) . XCL wason (XCL)  and sharon (XCL) . We can  effects .Artocarpus integrifolia, the peanut lectin from peanut Arachis hypogaea, and amaranth lectin from Amaranthus caudatus. These four lectins have been used in histochemistry for identification of the TF antigen in tissues [ABL is a member of a group of proteins, which bind the Thomsen-Friedenreich (TF) antigen selectively and with high affinity. TF antigen is represented by galactosyl β-1, 3-N-acetylgalactosamine and is common in malignant and pre-malignant epithelia ,11. Ther tissues ,13.Agaricus bisporus lectin (ABL), Arthrobotrys oligospora lectin, Xerocomus chrysenteron lectin (XCL), Pleurotus cornucopiae lectin, Gibberella zeae lectin, Paxilus involutus lectin [As previously reported by Rosen and al. , ABL bels lectin . The seqHere we focus on XCL binding constants for specific sugars and quantify the underlying thermodynamic parameters of the carbohydrate-XCL lectin interactions by direct measurement of the enthalpy using isothermal titration calorimetry method. We found that XCL recognizes TF antigen with high affinity (Kd: 1 μM).Red blood cell agglutination by XCL was inhibited when galactose, lactose and N-acetylgalactosamine was added to the system but no effect was seen with glucose, fucose, fructose, sorbitol, mannose and sucrose . We firs-1 and a reaction enthalpy of – 6.27 kcal/mol when the monomer concentration was considered for the calculations. As this affinity is very small, the enthalpy was estimated separately in another experiment where small quantities of lectin were injected into a N-acetylgalactosamine containing solution. We obtained a ΔH value of – 25.20 kcal/mole corresponding to four independent binding sites (data not shown).Figure -1 and no important change of the reaction enthalpy was observed . In many cases, binding of saccharides to lectins is coupled to changes in solvent accessibility that result in negative, albeit small, ΔCp values [Binding of carbohydrates to a number of proteins is characterised by small enthalpy and heat capacity changes. Hydrogen bonding interactions are essentially enthalpically driven with little change in the heat capacity, while hydrophobic interactions are essentially entropically driven . Measurep values . This is-1) and a reduction of the binding enthalpy  were observed. Thermodynamics parameters, which characterize the XCL – N-acetylgalactosamine interaction are summarised in Table Variation of osmotic stress allows to measure the energetic contribution of the solvatation effect on the enthalpy of the reaction . The wat-1 but with significant errors. These errors are due to an uncertainty in fitting the data at Ka values of smaller than 100 M-1.The N-acetyllactosamine was also used as a ligand and the affinity constant at 27°C is in the range of 50 M5 M-1 were obtained -D-GalNAc, since ABL was previously shown to bind this disaccharide with a high affinity constant. A ΔH value of -9.13 kcal/mole and an affinity constant of 1.06 10ned fig. . This vaFetuin and asialofetuin, which bear the TF antigen motif, were used to test XCL interactions with glycoproteins. Fetuin contains six oligosaccharides chains, namely three carbohydrate units O-linked to Thr or Ser residues and three complex glycans, N-linked to Asn residues . A fourt6 M-1) was found 4 times higher than for fetuin (5.9 105 M-1). This suggests that XCL binds asialofetuin more avidly than the native fetuin, and therefore that the presence of sialic acid reduces the affinity of XCL towards such glycans. The binding stoichiometry is of 0.23, which could correspond to 4 similar binding sites either on fetuin or asialofetuin. The binding enthalpy of XCL – fetuin/asialofetuin is of -21.5 kcal/mole and -16.8 kcal/mole respectively. This significant difference in the binding enthalpies of almost 5 kcal/mole leads us to conclude that in fetuin the sialic acids do contribute to the energy of binding.Several titrations of fetuin and asialofetuin to a XCL containing solution  were performed at 27°C. The binding isotherms for the titration of fetuin and asialofetuin into a XCL solution are presented in figure As we mentioned in the introduction, there are several TF-binding lectins. Althought they recognize the same motif, they have different actions on the proliferation phenomenon . For exaOn cell-surface glycoproteins, the epitope structure of TF antigen is α-linked to either serines or threonines . The aff6 M-1 for the asialofetuin-ABL interaction [6M-1). The XCL antiproliferative effect [At present, only limited informations on the thermodynamics datas of the lectin-sugar recognition are available and much work remains to be done to understand the underlying forces that govern these interactions. In this study, we investigate the specificity of XCL for carbohydrates and especially for Thomsen-Friedenreich antigen and glycoproteins bearing this disaccharide. Kinetic studies using a resonant mirror biosensor reported a binding affinity value of 3.3 10eraction  which ise effect  and the All products, mono- and di-saccharides, fetuin and asialofetuin from fetal bovine serum were purchased from sigma.E. coli BL21-DE3 strain. The histidine tag was added to facilitate the purification of the recombinant protein on an affinity column using nickel as ligand [600 nm of 1 was reached, the induction of T7 RNA polymerase with IPTG  was realized. Then the culture medium was allowed to grow overnight, at 16°C. Cells were harvested by centrifugation, washed and then lysed by sonication. Isolated XCL was purified by affinity chromatography on Ni-NTA column and dialyse methods. Protein purity was assessed using overloaded SDS-PAGE gels with Coomassie blue staining. XCL concentrations were determined spectrophotometrically from molar extinction coefficients at λ = 280 nm, ε = 31150.A fusion protein containing histidine tag, TEV site and XCL was expressed in s ligand  and the s ligand . Freshly2HPO4/NaH2PO4, pH = 7) from the last protein dialysis.Isothermal titration calorimetry was performed using a VP-ITC microcalorimeter from Microcal Inc. . Several experiments were performed to determine the binding constant values. In individual titrations, injections of 3 to 10 μl of carbohydrate/glycoprotein were added by computer-controlled 296 μl microsyringe at an interval of 200 seconds into the XCL solution (cell volume = 1.437 ml). The experiments were realized at 27°/10°C and a stirring speed of 300 rmp. 10% (w/v) PEG 8000 was used for some of the experiments. As the lectin affinity for sugars is relatively small, high sugar and protein concentrations were required. The XCL concentration varied between 0.14-0.4 mM, the monosaccharide concentrations between 30–50 mM, 0.95–3 mM for TF antigen and glycoproteins concentrations between 0.66–2 mM. The carbohydrates were dissolved in the buffer solution .a (association constant in M-1) and n (number of binding sites), as adjustable parameters. The monomer concentration was used throughout the analysis. The instrument was calibrated using the built in mode of electric field heat pulses. Thermodynamic parameters were calculated from the relation:The experimental data were fitted to a theoretical titration curve using software supplied by Microcal, with ΔH , KG = ΔH - TΔS = - RT ln KaΔ-1K-1.where the ΔG, ΔH and ΔS are the changes in free energy, enthalpy and entropy of binding; T is the absolute temperature and R = 1.98 cal molLD carried out the protein preparation and microcalorimetry work and drafted the manuscript. DF participated in the design of the study, MW participated in the microcalorimetry experiment and interpretations. LP conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.